1. Introduction

As the second largest ecological group of marine fungi, various chemical entities with unique structures and potent bioactivities which have high potential for exploitation in agriculture, medicine and industrial uses have been isolated from mangrove fungi [1, 2]. They are also suggested as promising sources for screening new natural products [2]. The tremulanes constitute a class of unusual sesquiterpenes that were first isolated from the aspen tree rotting fungus Phellinus tremulae in 1993 [3]. Thus far, more than 50 compounds of this class have been reported [4-9]. However, tremulane sesquiterpene lactones were rarely reported, and none of them have been isolated from mangrove endophytic fungi. In this paper, we report the isolation and structure elucidation of two new tremulane sesquiterpene lactones coriolopsin A (1) and coriolopsin B (2) , together with two known tremulane sesquiterpenes conocenol C (3) and ceriponol E (4) from a mangrove endophytic fungus, Coriolopsis sp. J5 (Fig. 1) . Herein the isolation, structure elucidation were reported.

2. Experimental 2.1. General

The NMR spectra were recorded on an AV-500 spectrometer (500 MHz for ¹H NMR and 125 MHz for ¹³C NMR; Bruker), using the solvent residue signal as the internal standard. Optical rotations were recorded using a Rudolph Autopol III polarimeter (Rudolph Research Analytical, Hackettstown, USA). The UV spectra were obtained from a DU-800 spectrometer (Beckman, Brea, USA). The IR spectra (KBr pellets) were run on a 380 FT-IR instrument from Nicolet (Thermo, Pittsburgh, USA). Column chromatography was performed with ODS gel (20-45 mm, Fuji Silysia Chemical Co., Ltd., Durham, USA), Sephadex LH-20 (Merck, Darmstadt, Germany) and silica gel (60-80, 200-300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). TLC was carried out on silica gel G precoated plates (Qingdao Haiyang Chemical Co., Ltd.), and spots were detected by spraying with 5% H₂SO₄ in EtOH followed by heating.

2.2. Fungal material

The mangrove endophytic fungus Coriolopsis sp. J5 was isolated from healthy branches of Ceriops tagal, collected from Dong Zhai Gang Mangrove National Nature Reserve in Hainan Province, China, in 2011. The fungus was identified by sequence analysis of the ITS region of its 18s rDNA. A BLAST search result indicated that the sequence was most similar (99%) to the sequence of Coriolopsis sp. (compared to AY336771.1) . The voucher sample was stored at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, and maintained on potato dextrose agar (PDA) slant at 4 ℃.

2.3. Fermentation, extraction, and isolation

The fungus stored on PDA medium was inoculated and cultured on PDA agar for 7 days. Seed medium (potato 200 g, dextrose 20 g, distilled water 1000 mL) in 500 mL × 10 Erlenmeyer flasks was inoculated with fungus and incubated at room temperature for 4 days on a rotating shaker (120 rpm). Production medium of solid rice in 1000 mL flasks (rice 50 g, distilled water 200 mL) was inoculated with seed solution (10 mL) one for one. Flasks were incubated at room temperature under static conditions and daylight for 60 days, cultures from 200 flasks were harvested for the isolation of substances. Following incubation, the mycelia and solid rice medium were extracted with EtOH through cheesecloth. Subsequently, the filtrate was extracted three times with an equal volume of Petroleum ether, EtOAc and n-BuOH, successively.

The EtOAc fraction (129.0 g) was chromatographed on a silica gel column using a step gradient elution of CHCl₃-MeOH (1:0-0:1, v/v) to afford ten fractions (Fr. 1-Fr. 10) . Fr. 5 (9.9 g) was applied to octadecyl silane (ODS) gel with gradient elution of MeOH-H₂O (3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 1:0) to yield 19 subfractions (Fr. 5-1-Fr. 5-19) . Subfraction Fr. 5-7 (354.2 mg) was chromatographed on a Sephadex LH-20 column using CHCl₃/MeOH (1:1) and then chromatographed on silica gel column with gradient elution of Petroleum ether-EtOAc (20:1, 18:1, 15:1, 11:1) to give compound 3 (4.8 mg). Subfraction Fr. 5-8 (411.0 mg) was chromatographed on silica gel column with gradient elution of Petroleum ether- EtOAc (15:1, 11:1, 10:1) and then chromatographed on a Sephadex LH-20 column using acetone to give compound 2 (6.8 mg). Fr. 7 (20.0 g) was applied to octadecyl silane (ODS) gel with gradient elution of MeOH-H₂O (3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 1:0) to yield 12 subfractions (Fr. 7-1-Fr. 7-12) . Subfraction Fr. 7-2 (255.8 mg) was chromatographed on silica gel column with gradient elution of Petroleum ether-EtOAc (5:1) and then chromatographed on a Sephadex LH-20 column using acetone to give compound 4 (8.8 mg). Subfraction Fr. 7-9 (346.5 mg) was chromatographed on silica gel column with gradient elution of Petroleum ether-EtOAc (15:1) and then chromatographed on a Sephadex LH-20 column using acetone to give compound 1 (21.3 mg).

Coriolopsin A (1) Colorless needle crystal; m.p. 192.1-193.7 ℃; [α]D²⁵ +87.6 (c 0.05, MeOH); UV (MeOH) λ_max (log e): 239 (3.74) nm; IR (KBr) ν_max (cm^-1) : 2923, 1645 (C55O), 1378, 1037, 1039; For ¹H NMR and ¹³C NMR spectral data, see Table 1; HR-ESIMS m/z: [M+H]⁺ 249.1483 (calcd. for 249.1485) .

Coriolopsin B (2) Colorless oil; [a]D 25 -21.8 (c 0.05, MeOH); UV (MeOH) lmax (log e): 240 (3.21) nm; IR (KBr) nmax (cm^-1) : 3402, 2925, 1632 (C55O), 1384, 1053; For ¹H and ¹³C NMR spectral data, see Table 1; HR-ESIMS m/z: [M+Na]+ 287.1253 (calcd. for 287.1254) .

Crystal data of coriolopsin A (1) : Colorless crystals of 1 were obtained in acetone-MeOH (10: 1, v/v). The data collection was acquired on a Bruker D8 QUEST diffractometer (Bruker). All intensity measurements were performed using graphite monochromated Cu Ka radiation (l = 0.71073Å ). In monoclinic, space group P21/n, unit cell dimensions a = 8.4525 (14) Å , b = 6.2926 (10) Å , c = 12.938 (2) Å , α = 90°, β = 103.044 (7) 8, γ = 90°, V = 670.39 (19) Å , Z = 2, Dx = 1.230 g/cm^-3, F(0 0 0) = 268.0, m = 0.678 mm^-1, the final R = 0.0629 and wR = 0.1464 for 2351 observed reflections [I > 2σ (I)] (see Fig. 3) . Crystallographic information file of Compound 1 with Cambridge Crystallographic Data Centre (CCDC) reference number 1459000 has been deposited at the CCDC, and can be obtained free of charge from the CCDC via https://www.ccdc.cam.ac.uk/deposit/.

2.4. Biological assays

Cytotoxic activity was tested by the MTT method as described previously [10]. Three cancer cell lines, Human chronic myelogenous leukemia cell line (K562) , human gastric carcinoma cell line (SGC-7901) , and human hepatocellular carcinoma (BEL-7402) were used. Antibacterial activity was determined by the filter paper disc diffusion method against Staphylococcus aureus (ATCC51650) , Ralstonia solanacearum, Fusarium oxysporum f.sp. cubense race 4, Fusarium oxysporum f.sp. niveum, Fusarium oxysporum f.sp.vasinfectum and Candida albicans (ATCC 10231) [11].

3. Results and discussion

Compound 1 was obtained as colorless needle crystal. The molecular formula of 1 was deduced as C₁₅H₂₀O₃ by HR-ESIMS at m/z 249.1483 [M+H]⁺ (calcd. for C₁₅H₂₀O₃, 249.1485) and ¹³C NMR spectral data, corresponding to six degrees of unsaturation. This was corroborated by the ¹³C NMR and DEPT NMR spectra, which displayed 15 resonances for one sp³ quaternary carbon (δ_C 39.1) , two disubstituted olefinic carbons (δ_C 169.9 and 120.9) , three methines (δ_C 51.4, 46.9 and 32.5) , four methylenes (δ_C 49.6, 44.6, 36.8 and 22.5) , three methyls (δ_C 28.5, 27.2 and 12.1) , and two ester carbonyls (δ_C 173.8 and 164.4) . These observations, in combination with the molecular formula, revealed that 1 possessed three rings. The above-mentioned data exhibited similarities with those of Tremulenediol A [3], except that two ester carbonyls signals of 1 replace the corresponding two hydroxymethyl signals of Tremulenediol A. ¹H-¹H COSY correlations of H-3/H-4/H-5/H-6/H-7/H-8 and H-6/H-13, and HMBC correlations from H₃-14 (δ_H 0.88) and H₃-15 (δ_H 1.18) to C-8 (δ_C 44.6) , C-10 (δ_C 49.6) and C-9 (δ_C 39.1) , from H₂-10 (δ_H 3.13 and 2.18) , H1-7 (δ_H 3.21) and H1-3 (δ_H 3.78) to C-1 (δ_C 169.9) and C-2 (δ_C 120.9) , from H₂-4 (δ_H 1.99 and 1.86) to C- 2 (δ_C 120.9) , corroborated the existence of the C-1-C-10 substructure and the positions of CH₃-13 (δ_C/H 12.1/0.89) , CH₃- 14 (δ_C/H 27.2/0.88) , and CH₃-15 (δ_C/H 28.5/1.18) . The presence of the dihydrofuran-2, 5-dione moiety was deduced by the molecular formula in combination with the key HMBC correlations from H-3 (δ_H 3.78) to C-11 (δ_C 164.4) and C-12 (δ_C 173.8) (see Fig. 2) . Finally, [M+Na]⁺ 1 was determined by single crystal X-ray diffraction analysis (see Fig. 3) . The final refinement on the Cu Ka data resulted in a Flack parameter of -0.03 (11) , allowing an unambiguous assignment of the complete absolute configurations of all the chiral centers as 3S, 6R, and 7R.

Compound 2, obtained as colorless oil and was assigned a molecular formula of C₁₅H₂₀O₄, as determined by HR-ESIMS (found [M+Na]⁺ 287.1253, calcd. for 287.1254) and nuclear magnetic resonance (NMR) data, corresponding to six degrees of unsaturation. This was corroborated by the ¹³C NMR and DEPT NMR spectra, which displayed 15 resonances for one ester carbonyl (δ_C 176.4) , one carboxylic carbonyl carbon (δ_C 169.3) , two disubstituted olefinic carbons (δ_C 121.7 and 160.2) , two sp³ quaternary carbons (δ_C 35.9 and 82.6) (where one was oxygenated), two allylic sp³ methines (δ_C 57.5 and 41.4) , four endo-cyclic methylenes (δ_C 49.7, 43.9, 26.8 and 26.1) , and three methyls (δ_C 30.6, 29.4 and 28.7) . These observations, in combination with the molecular formula, revealed that 2 possessed three rings. Comparison of NMR data between 1 and 2 suggested that their structures were closely related. The main difference was that the CH-6 in 1 was replaced by an oxygenated quaternary carbon in 2. These data, together with that an additional O atom was present in the molecular formula of 2 compared with that of 1, indicated that the dihydrofuran-2, 5-dione moiety in 2 was hydrolyzed and then the -COOH at C-3 (δ_C/H 41.4/3.82) was linked with C-6 (δ_C 82.6) to afford an ester bridge. HMBC and ¹H-¹H COSY data as shown in Fig. 2 supported this assignment. In the ROESY spectrum, correlations of H-7 (δ_H 3.20) with H₃-14 (δ_H 1.08) and H₃-13 (δ_H 1.38) , were observed, suggesting that these protons were on the same face, and consequently the ester bridge was on the other face (see Fig. 4) .

The structures of compounds 3-4, which have been isolated from Coriolopsis sp. J5, were identified by comparison of their spectroscopic data with those reported in the literatures as conocenol C (3) [4] and ceriponol E (4) [7]. All compounds were isolated from the fermentation extract of endophytic fungus Coriolopsis sp. J5 from Ceriops tagal for the first time.

All compounds were evaluated for cytotoxic activities against three human tumour cell lines (K562, SGC-7901, and BEL-7402) and antibacterial activities against Staphylococcus aureus (ATCC51650) , Ralstonia solanacearum, Fusarium oxysporum f. sp. cubense race 4, Fusarium oxysporum f.sp. niveum, Fusarium oxysporum f.sp. vasinfectum and Candida albicans (ATCC10231) . However, none of the compounds showed obvious cytotoxic or antibacterial activities.

4. Conclusion

Two new tremulane sesquiterpenes coriolopsin A (1) and coriolopsin B (2), along with two known ones conocenol C (3) and ceriponol E (4), were isolated from the fermentation extract of endophytic fungus Coriolopsis sp. J5 from Ceriops tagal. The structures of 1-4 were elucidated by NMR experiments with the aid of X-ray diffraction analysis. In the bioassay, none of the compounds showed obvious cytotoxic or antibacterial activities.

Acknowledgments

This research was financially supported by National Natural Science Foundation of China (Nos. 41406083, 41506096) and National Nonprofit Institute Research Grant of CATAS-ITBB (No.ITBB2015ZY04, ITBB2015RC02) .